Cytochrome c films formed on silane self-assembled monolayer derivatized surfaces

Abstract

Formation and characterization of well-organized protein film assemblies are of high interest due to potential applications in biomolecular devices. The hypothesis that a macroscopically ordered protein film can be formed by site-directed, covalent binding of a protein to an appropriately derivatized surface formed the basis for the reported studies. The molecular architecture chosen to address this hypothesis consisted of yeast cytochrome c, a heme protein containing a unique binding site, immobilized on a surface derivatized with a silane self-assembled monolayer, (SAM). A combination of two techniques: total internal reflectance fluorescence (TIRF), to measure fluorescence anisotropy, and integrated optical waveguide-attenuated total reflectance (IOW-ATR) spectroscopy, to measure absorbance linear dichroism, was used to probe the macroscopic order of the heme groups in the film assemblies. Epifluorescence microscopy and absorbance (in an IOW-ATR geometry) were used to probe the nature of the surface-protein interactions and to determine relative protein affinities for different SAM-derivatized surfaces. The molecular orientation distribution for yeast cytochrome c immobilized on a 100% thiol-terminated SAM was 67° ± 39°. Partial protein removal was observed under a variety of rinse conditions, indicating that multiple protein-surface interactions may have contributed to the wide distribution value. Relative binding affinity constants and protein-surface interactions are compared for yeast cytochrome c and horse heart cytochrome c on 100% thiol-capped, 100% hydroxyl-capped, and mixed SAM-modified surfaces. These studies were also extended to include a variant of yeast cytochrome c, Thr8Cys/Cys102Thr. Similar adsorption and removal trends were observed for all the protein-SAM combinations. The adsorption isotherms indicated that at least two binding processes occur during formation of each protein film, high and low affinity binding. Removal studies indicated that the adsorption process is only partially reversible. It was concluded that employing a site-directed immobilization strategy does not necessarily produce a well-ordered protein film.